The gene for Werner's syndrome, an adult-onset progeroid syndrome, has recently been identified by positional cloning. This approach cannot be used to clone the Hutchinson-Gilford Progeria Syndrome (HGPS) gene because there are no families with HGPS pedigrees. It is therefore proposed to clone the HGPS gene by functional complementation. To clone a gene by functional complementation it is critical to identify a reliable, measurable, phenotypic marker in the diseased cells that is not present in normal cells. But so far, no reliable cellular phenotype has been identified in HGPS. We have recently identified several genes whose expression is markedly upregulated in HGPS cells by expression profiling using high-density oligonucleotide microarrays ("GeneChips"). These genes can now serve as phenotypic markers unique to HGPS fibroblasts. To test the widely held hypothesis that HGPS is the result of an autosomal dominant mutation, HGPS and normal fibroblasts will be fused, and the gene expression profile of the hybrid cells will be analyzed by microarrays. These experiments will also identify those markers that can be used in further studies, namely those genes for which the abnormal level of expression characteristic of HGPS fibroblasts is maintained in the HGPS/normal hybrid lines. To prepare and test cell lines in which the expression of the abnormally expressed genes can be assayed at the single cell level, the promoter regions of the selected marker genes will be fused with reporter genes assayable by fluorescence (such as the green fluorescent protein, or GFP), and stably transfected into normal fibroblasts. Cell lines will be selected in which the expression of the GFP reporter gene(s) mirrors exactly the expression of the endogenous gene(s). To clone the HGPS gene, high titer retroviral cDNA libraries prepared from HGPS cells will be used to infect the cell lines carrying the reporter constructs. Pools of infected cells will be analyzed by flow cytometry and cell sorting to identify cells in which reporter gene expression becomes characteristic of an HGPS/normal hybrid rather than of the normal cell line. Once the putative HGPS gene is identified in this manner, the complete sequence of the gene will be determined in normal and HGPS cells to ascertain the nature of the mutational change.